Cell-specific targeting for delivery of effector moieties such as diagnostic or therapeutic agents is a widely researched field and has led to the development of non-invasive diagnostic and/or therapeutic medical applications. In particular in the field of nuclear medicine procedures and treatments, which employ radioactive materials emitting electromagnetic radiations as γ-rays or photons or particle emitting radiation (e.g. alpha, beta, Auger-electrons), selective localization of these radioactive materials in targeted cells or tissues is required to achieve either high signal intensity for visualization of specific tissues, assessing a disease and/or monitoring effects of therapeutic treatments, or high radiation dose, for delivering adequate doses of ionizing radiation to a specified diseased site, without the risk of radiation injury in other e.g. healthy tissues. It is thus of crucial interest to determine and assess cell-specific and in particular tumour-selective structures such as receptors, antigens and the like which can be specifically targeted by the respective biological vehicles.
The folate receptor (FR) has been identified as one of these structures. The FR is a high-affinity (KD<10−9 M) membrane-associated protein. In normal tissues and organs FR-expression is highly restricted to only a few organs (e.g. kidney, lungs, choroids plexus, and placenta), where it largely occurs at the luminal surface of epithelial cells and is therefore not supplied with folate in the circulation. The FR-alpha is frequently overexpressed on a wide variety of specific cell types, such as epithelial tumours (e.g. ovarian, cervical, endometrial, breast, colorectal, kidney, lung, nasopharyngeal), whereas the FR-beta is frequently overexpressed in leukaemia cells (approx. 70% of acute myelogenous leukaemia (AML) are FR-beta positive). Both may therefore be used as a valuable tumour marker for selective tumour-targeting (Elnakat and Ratnam, Adv. Drug Deliv. Rev. 2004; 56:1067-84). In addition it has recently been discovered that activated (but not resting) synovial macrophages in patients diagnosed with rheumatoid arthritis possess a functionally active FR-beta. Therefore activated macrophages can be selectively targeted with folate conjugates in arthritic joints, a capability that opens possibilities for the diagnosis and treatment of rheumatoid arthritis (Paulos et al, Adv. Drug Deliv. Rev. 2004; 56:1205-17). Folic acid and its derivatives have thus been intensively studied over the past 15 years as targeting agents for the delivery of therapeutic and/or diagnostic agents to cell populations bearing folate receptors in order to achieve a selective concentration of therapeutic and/or diagnostic agents in such cells relative to normal cells. Various probes have been conjugated to folic acid and (pre)clinically evaluated, including folate radiopharmaceuticals (Leamon and Low, Drug Discov. Today 2001; 6:44-51), folate-conjugates of chemotherapeutic agents (Leamon and Reddy, Adv. Drug Deliv. Rev. 2004; 56:1127-41; Leamon et al, Bioconjugate Chem. 2005; 16:803-11), proteins and protein toxins (Ward et al., J. Drug Target. 2000; 8:119-23; Leamon et al, J. Biol. Chem. 1993; 268:24847-54; Leamon and Low, J. Drug Target. 1994; 2:101-12), antisense oliconucleotides (Li et al, Pharm. Res. 1998; 15:1540-45; Zhao and Lee, Adv. Drug Deliv. Rev. 2004; 56:1193-204), liposomes Lee and Low, Biochim. Biophys. Acta-Biomembr. 1995; 1233:134-44; Gabizon et al, Adv. Drug Deliv. Rev. 2004; 56:1177-92), MRI contrast agents (Konda et al, Magn. Reson. Mat. Phys. Biol. Med. 2001; 12:104-13) etc.
However, apart from specific uptake of folate-conjugates in tumours, substantial and specific accumulation was also observed in kidneys because of considerable expression of FRs in proximal tubule cells of the renal tissue (Mathias et al, J. Nucl. Med. 1998, 39:1579-85; Mathias and Green, Nucl. Med. Biol. 1998, 25:585-87; Mathias and Green, J. Nucl. Med. 2000, 41:1113; Siegel et al, J. Nucl. Med. 2003, 44:700-07; Leamon et al, Bioconjugate Chem. 2002, 13:1200-10; Reddy et al, J. Nucl. Med. 2004, 45:857-66; Mathias et al, Bioconjugate Chem. 2000, 11:253-57; Trump et al, Nucl. Med. Biol. 2002, 29:569-73; Wang et al, Bioconjugate Chem. 1997, 8:673-79). This issue severely limit the use of currently known folate-mediated targeting methods in various medical applications and in particular the use of therapeutic folate/pteroate-conjugates radiolabeled with particle-emitting radioisotopes (β− or α), since application of high radioactivity in order to achieve a cytotoxic radiation dose in tumour cells would consequently comprise the risk of radiation injury in sensitive renal tissue. Therefore, FR tumour-targeting has not been exploited for its use in radiotherapy so far.
In view of the above limitations, there is clearly a need for appropriate targeting methods which show high selectivity of the folate/pteroate-conjugates to the targeted sites and efficiency (and thus adequate safety) for their use in various medical applications.
Applicants have now found that these disadvantages can be overcome by simultaneous or sequential administration of antifolates and folate/pteroate-conjugates, which led to a drastic increase in tumour selectivity of the folate-based radiopharmaceutical (i.e. increase of the tumour-to-kidney ratio of radioactivity).